Bulkiness of Surface Ligands to Promote the Interaction of

Jan 21, 2016 - ... sufficiently strong to cause overt disruption of lipid vesicles and cell membranes. ... liposomes are also substantially more disru...
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Aromaticity/Bulkiness of Surface Ligands to Promote the Interaction of Anionic Amphiphilic Gold Nanoparticles with Lipid Bilayers Jinhong Gao, Ouyang Zhang, Jing Ren, Chuanliu Wu,* and Yibing Zhao* The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, P.R. China S Supporting Information *

ABSTRACT: The presence of large hydrophobic aromatic residues in cell-penetrating peptides or proteins has been demonstrated to be advantageous for their cell penetration. This phenomenon has also been observed when AuNPs were modified with peptides containing aromatic amino acids. However, it is still not clear how the presence of hydrophobic and aromatic groups on the surface of anionic AuNPs affects their interaction with lipid bilayers. Here, we studied the interaction of a range of anionic amphiphilic AuNPs coated by different combinations of hydrophobic and anionic ligands with four different types of synthetic lipid vesicles. Our results demonstrated the important role of the surface aromatic or bulky groups, relative to the hydrocarbon chains, in the interaction of anionic AuNPs with lipid bilayers. Hydrophobic interaction itself arising from the insertion of aromatic/ bulky ligands on the surface of AuNPs into lipid bilayers is sufficiently strong to cause overt disruption of lipid vesicles and cell membranes. Moreover, by comparing the results obtained from AuNPs coated with aromatic ligands and cyclohexyl ligands lacking aromaticity respectively, we demonstrated that the bulkiness of the terminal groups in hydrophobic ligands instead of the aromatic character might be more important to the interaction of AuNPs with lipid bilayers. Finally, we further correlated the observation on model liposomes with that on cell membranes, demonstrating that AuNPs that are more disruptive to the more negatively charged liposomes are also substantially more disruptive to cell membranes. In addition, our results revealed that certain cellular membrane domains that are more susceptible to disruption caused by hydrophobic interactions with nanoparticle surfaces might determine the threshold of AuNP-mediated cytotoxicity.

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INTRODUCTION Enormous advances have been made in the field of nanoparticle synthesis and surface modification, which facilitate the fabrication of sophisticated nanostructures for diverse biomedicine applications, such as drug delivery, bioimaging, and cancer therapy.1−5 For most of these applications, nanoparticles have to overcome cellular membrane barriers through endocytosis and/or spontaneous penetration to enter into the cytosol.6−10 An essential step for the entry is that nanoparticles adhere to and interact with cellular membranes.11−13 Therefore, a profound understanding of this essential step is important for the better tailoring and handling of nanoparticles used in biological systems. Many studies have been conducted in the past decade, using various nanoparticles and membrane models, to investigate nanoparticle−membrane interactions.14−20 The adhesion properties of nanoparticles to lipid membranes are determined by many factors including the size,21−23 shape,18 and surface property14,21,24,25 of nanoparticles. In particular, surface properties of nanoparticles such as charge,26 hydrophobicity,21 and surface defects27 can dramatically affect the adhesion properties of nanoparticles and their interaction behaviors, which © XXXX American Chemical Society

ultimately determine their cellular uptake pathway. Take monolayer-protected gold nanoparticles (AuNPs) for example: cationic AuNPs display more intensive adhesion to the cell membranes, relative to their anionic partners, which leads to higher efficiency of cellular uptake.1,28,29 Moreover, the cationic surface of AuNPs can transiently disrupt the integrity of cellular membrane bilayers, and consequently result in spontaneous penetration of Au NPs into the cytosol.9,10 A primary driver for this process comes from the strong electrostatic attraction between nanoparticles and membranes. This uptake pathway is also applied to other cationic nanoparticles. However, the disruptive nanoparticle−membrane interactions associate, at least partially, with cytotoxicity, which to some extent limits the biomedicine applications of cationic nanoparticles.8,26,30 More recently, researchers found that anionic AuNPs with structured surfaces composed of a binary mixture of hydrophobic and anionic ligands can nondisruptively penetrate cellular membranes, involving a pathway fundamentally different from that Received: January 5, 2016 Revised: January 18, 2016

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Figure 1. (a) Chemical structures of the diverse surface ligands on the surface of AuNPs. (b) Chemical structures of lipids and cholesterol used for the preparation of four different types of liposomes.

Figure 2. (a) A representative TEM image of AuNPs. (b) The diameter distribution histogram of AuNPs. (c) UV−vis absorption spectra of MUAAuNPs, C16-AuNPs and C11-Trp-AuNPs (∼2.4 nM) in water (10 mM aqueous tetramethylammonium hydroxide solution). (d) CTAT titration indicating the different surface properties of the three AuNPs.

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Figure 3. (a) Cartoon illustrating the fluorescence dequenching resulting from the leakage of dyes from the destabilized liposome. (b) AuNPs induced time-dependent CF leakage from DOPC liposomes in 20 mM HEPES buffer; [lipid] = 2.3 μM, [AuNPs] = 0.2 nM. (c) Size distributions for mixtures of AuNPs and DOPC liposomes determined by DLS after 1 h incubation; [lipid] = 6.9 μM, [AuNPs] = 0 (black line, the control), 6 nM (red and blue line, MUA-AuNPs and C11-Trp-AuNPs, respectively), 0.6 nM (green line, C16-AuNPs). (d) AuNPs induced time-dependent CF leakage from DOPC/Chol liposomes in 20 mM HEPES buffer; [lipid] = 2.3 μM, [AuNPs] = 0.2 nM. Data are presented as mean ± s.d., n = 3.

of cationic nanoparticles.29,31,32 The ribbon-like distribution of ligands on the surface of Au NPs has been demonstrated to be vital for the nondisruptive penetration of anionic AuNPs through membranes. The nondisruptive interaction of anionic AuNPs with cellular membranes has been considered a spectacle that resembles the penetration of some cell-penetrating peptides with ordered and amphiphilic structure.33−35 The fusion of anionic AuNPs with membranes is also considered similar to transmembrane proteins that nondisruptively reside within lipid bilayers.36−38 In both cell-penetrating peptides and transmembrane proteins, aromatic residues, especially tryptophan (Trp), play a crucial role in mediating their interactions with cellular membranes.39−41 Moreover, the presence of tryptophan residues in cell-penetrating peptides has frequently been demonstrated to be advantageous for their cellular uptake;42,43 also recently, Yang et al. showed that the surface tryptophan-bearing peptide ligands can significantly enhance the cellular uptake of anionic AuNPs.44 Despite this, as a promising synthetic analogue of these biomacromolecules, anionic AuNPs that were used for mechanism study of the nanoparticle−membrane interactions usually bear no aromatic groups on their surface. With these in mind, it would be interesting to explore how the incorporation of aromatic group-containing ligands onto the surface of anionic AuNPs affects their interaction with lipid bilayers. To the best of our knowledge, studies on this are still lacking. In this work, we explore the interaction of anionic amphiphilic AuNPs with synthetic lipid vesicles (i.e., liposomes). AuNPs with an identical diameter (∼5 nm), coated by a monolayer of hydrophobic and anionic ligands with a thiol-end group, were prepared by a ligand-exchange approach. The length of the hydrophobic ligands and their terminal aromatic groups were systematically changed to tune the

surface amphiphilic property of anionic AuNPs, which generates ∼12 different AuNPs (Figure 1a). Four different types of lipid vesicles were used as model bilayers for mechanism study of nanoparticle−membrane interactions (Figure 1b). Our results reveal the important role of the surface bulky hydrophobic groups, relative to hydrocarbon chains, in the adhesion and interaction of anionic AuNPs with lipid bilayers.

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RESULTS AND DISCUSSION Three types of monolayer-protected AuNPs were first prepared and studied (Figure 1a). That is, AuNPs protected by (1) 11mercaptoundecanoic acid (MUA) (MUA-AuNPs), (2) a mixture of MUA and tryptamine-conjugated MUA (referred to as C11-Trp) (C11-Trp-AuNPs), and (3) a mixture of MUA and 1-hexadecannethiol (HT or C16) (C16-AuNPs), respectively. These AuNPs were obtained from a ligand exchange of the dodecylamine-coated AuNPs with MUA or a 1:1 molar mixture of the binary ligands, and thus possess an identical core diameter. AuNPs were characterized by transmission electron microscopy (TEM), indicating a narrow distribution of the diameter (5.2 ± 0.6 nm, Figure 2a,b). The dodecylamine ligands weakly bound to AuNPs can be easily displaced by thiol-bearing ligands, and UV−vis absorption spectra demonstrate the preservation of nanoparticle monodispersity after the ligand exchange (Figure 2c). All these anionic AuNPs are water-soluble and display good stability in aqueous solutions. No obvious aggregation was observed after 3 months of storage at 4 °C in 10 mM aqueous tetramethylammonium hydroxide solution. For C16-AuNPs, despite the absence of obvious change in the UV−vis absorption, partial aggregation in solution was still observed by dynamic light scattering (DLS) (Figure S1). However, electrostatic repulsion between the C

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To compare the affinity of C16-AuNPs and C11-Trp-AuNPs toward DOPC liposomes, a fluorescence quenching assay was then developed. We first prepared DOPC liposomes doped with 0.1% L-α-phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) (POPE-RhB, a rhodamine B-conjugated lipid), using the same procedure for the preparation of DOPC liposomes. Then, the fluorescence of rhodamine B on the surface of the liposomes was monitored upon the addition of the AuNPs. For the sake of higher quench efficiency of the fluorescence of POPE-RhB by AuNPs, a 10-fold higher ratio of AuNPs/liposomes (∼30:1) than that in leakage assays was used, which would lead to the formation of a monolayer of AuNPs on the outer leaflet of the lipid membrane if all AuNPs can be adsorbed onto the surface. We found that the incubation with C16-AuNPs can result in a substantially more obvious quenching of fluorescence (∼50% quenched, considering that only POPE-RhB on the outer leaflet can be quenched) compared to either MUA-AuNPs or C11-Trp-AuNPs (Figure S5), suggesting that C16-AuNPs exhibit a relatively higher affinity toward DOPC liposomes. The negligible quenching of fluorescence by C11-Trp-AuNPs suggests that their interactions with DOPC liposomes were transient or too weak to be monitored by the assay. To further explore the mechanism of nanoparticle-induced liposome leakage, DLS was used to probe the diameter change of liposomes upon incubation with AuNPs. As seen in Figure 3b, incubation with C16-AuNPs results in a significant increase in hydrodynamic diameters, suggesting the rapid aggregation and/or fusion of DOPC liposomes, consistent with the burstrelease kinetics observed in Figure 3a. By contrast, litter change in diameter was observed upon incubation with MUA-AuNPs or C11-Trp-AuNPs (Figure 3c), though their concentrations were much higher than that of C 16 -AuNPs and the concentration used for the CF-leakage experiment. We further calculated the octanol−water partition coefficient of the ligands (C16 and C11-Trp) using Interactive logP Calculator. The result indicates that the surface ligand C16 is obviously more hydrophobic than C11-Trp (log P: 8.12 and 5.77 for C16 and C11-Trp, respectively), which is in agreement with the CTAT titration characterizations, strongly suggesting that the disruption effect of the C16-AuNPs on the liposome integrity should be caused by the strong interface hydrophobic interactions. In contrast, indole rings on the surface of AuNPs can only cause the permeation of the DOPC bilayers without severe destruction of liposome entity due to, to some extent at least, the relatively low hydrophobicity. Lipid rafts in cell membranes play important roles in regulation of cell functions, into which small molecules such as cholesterol (Chol) are incorporated. The presence of Chol within lipid bilayers can fill voids created by interdigitating fatty acid chains and optimize membrane packing.20,47 In addition, the filled Chol molecules can facilitate the association of proteins or synthetic nanomaterials to cell membranes through hydrophobic interactions.48 Accordingly, we then synthesized Chol-filled DOPC liposomes (DOPC/Chol liposomes with DOPC/Chol = 2:1) containing self-quenched CF dyes (Figure 1b), and evaluated their adhesion and interaction with the above anionic AuNPs by leakage assays. The hydrodynamic diameter and ζ-potential of DOPC/Chol liposomes were determined to be 133 nm (Figure S6) and −12.2 mV, respectively, similar to that of DOPC liposomes. Figure 3d shows the time-dependent liposome leakage upon incubation with AuNPs. A summary table describing outcomes of the

negatively charged AuNPs prevented their further overt aggregation. DLS measurements further indicate that C11Trp-AuNPs and MUA-AuNPs are both monodisperse in solution (hydrodynamic diameter: ∼ 10 nm; see Figure S1). However, further increase in the molar ratio of the C11-Trp in the process of ligand exchange resulted in severe aggregation of AuNPs in water because of the increased surface hydrophobicity, as evidenced by UV−vis absorption (Figure S2). The surface property of these anionic AuNPs was then characterized by titration with a cationic surfactant cetyltrimethylammonium tosylate (CTAT).31 Stepwise addition of 1.0 mM CTAT solution to 2.4 nM MUA-AuNPs caused spectroscopically visible aggregation of nanoparticles at the point of overall charge neutrality, monitored by the ratio of absorbance at 650 and 520 nm (Figure 2d). The aggregation point of C11-Trp-AuNPs and C16-AuNPs is respectively later and earlier than that of MUA-AuNPs, indicating the difference in their surface properties. Particularly, C11-Trp-AuNPs were significantly more tolerant to the addition of CTAT than other two AuNPs, not only in terms of the CTAT content, but also the degree of aggregation, further indicating the unique anionic and amphiphilic properties of the C11-Trp-AuNPs. Prior aggregation of C16-AuNPs relative to MUA-AuNPs caused by CTAT reflects, as expected, the decrease of negative charges on a nanoparticle surface as part of MUA ligands were displaced by HT.31 In addition, we determined the surface composition of the two anionic AuNPs using 1H NMR (see the Supporting Information, Figure S3). We found that their surfaces contain a comparable fraction of hydrophobic ligands (7.4% and 9.1% for C11-Trp-AuNPs and C16-AuNPs, respectively), though a molar ratio of 1:1 (MUA and hydrophobic ligand) was used for the ligand exchanges. To explore the interaction of these AuNPs with a membrane, we then prepared lipid vesicles (or liposome) from DOPC (1,2dioleoyl-sn-glycero-3-phosphocholine) by using a reported extrusion method,43,45 into which carboxyfluorescein (CF) was encapsulated at a high concentration (75 mM). As the fluorescence of the encapsulated CF dyes was self-quenched, small perturbations on the membranes by the adhesion and interaction of external materials can lead to liposome leakage and subsequent recovery of fluorescence (Figure 3a). This response provides a facile and sensitive assay to probe the adhesion and interaction of nanoparticles with lipid bilayers.45,46 The as-prepared DOPC liposomes have a hydrodynamic diameter of 131 nm (Figure S4) and a ζ-potential of −14.7 mV. Figure 3b shows the kinetics curves of liposomeleakage upon incubation with C16-AuNPs, C11-Trp-AuNPs, and MUA-AuNPs, respectively, and a control curve for only liposomes present. C16-AuNPs induce a very rapid leakage that completes the release of CF after ∼50 min. By contrast, the liposome leakage induced by C11-Trp-AuNPs is relatively moderate, and the release gradually increases to ∼80% after 6 h. MUA-AuNPs only induce negligible leakage, which is very similar to the control sample. These results first indicate the strong hydrophobic binding of C16-AuNPs and DOPC liposomes, which is in agreement with a previous report,31 second, demonstrate that surface aromatic and bulky indole ring groups (C11-Trp) can efficiently induce the adhesion and interaction of AuNPs with DOPC bilayers, and, third, indicate that the disrupting effect of the surface hydrocarbon chains to the membrane integrality can be significantly weakened by replacing with the aromatic indole rings. D

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can efficiently prevent the adhesion and interaction of AuNPs mediated by the hydrophobic hydrocarbon chains, but not the aromatic and bulky indole rings, though the binding of C11-TrpAuNPs to DOCP liposomes (relative to DOPC liposomes) is relatively weak because of the increased electrostatic repulsions, as evidenced by the observation of DLS signal from free AuNPs and fluorescence quenching assays (Figure S8). This finding is particularly interesting considering the fact that some domains of cell membranes are significantly more negatively charged than DOPC bilayers due to the presence of negatively charged carbohydrate coats.48,50 Considering that the aromatic indole ring structure in C11Trp is significantly bulkier than hydrocarbon chains and, as a consequence, has more chances of exposure to solution and adhesion to membranes, we further examined the effect of the length of hydrophobic ligands, relative to the anionic MUA, on the membrane−AuNPs interactions. Two tryptamine-conjugating ligands, C6-Trp and C3-Trp, which are shorter than C11-Trp in length, but comparable to or shorter than MUA, respectively, were designed and synthesized (Figure 1a). Three new types of AuNPs protected by a mixture of (1) MUA and C6-Trp (C6Trp-AuNPs), (2) MUA and C3-Trp (C3-Trp-AuNPs), and (3) MUA and 1-octanethiol (OT or C8) (C8−AuNPs) respectively were then prepared by ligand exchanges. These AuNPs were characterized by UV−vis absorption (Figure S9), confirming that all of them have good stability and monodispersity in aqueous solutions. In addition, CTAT titration characterizations indicate that AuNPs protected by a mixture of MUA and tryptamine-capping ligands display similar surface properties, and C8-AuNPs resemble C16-AuNPs or MUA-AuNPs (Figure S10), thus favoring the evaluation of the effect of ligand length reliably and definitely. Figure 5a−c shows the leakage of dyes from the three different liposomes during incubation with C6-Trp-AuNPs, C3-Trp-AuNPs, and C8-AuNPs, respectively. Interestingly, we found that C6-Trp-AuNPs and C3-Trp-AuNPs induce leakage of CF dyes from liposomes (DOCP and DOPC) following a kinetic process that is quite similar to that of C11-Trp-AuNPs (Figure 5a and 5c). Likewise, the dye leakage from these two types of liposomes can be prevented by the incorporation of Chol molecules, which increases the compactness of membrane bilayers (Figures 5b and S11). By contrast, a decrease in the length of the hydrocarbon chains (HT versus OT) results in a “block shot” of OT by MUA ligands on the surface of C8−AuNPs, which subsequently eliminates the disrupting effect of the surface-exposure hydrocarbon groups to the membrane integrality (Figure 5a−

interaction of AuNPs with liposomes was given in Table 1. Similar to the pure DOPC system, C16-AuNPs can still Table 1. A Summary of Results on the Leakage of CF Resulting from the Interaction of AuNPs with Liposomesa liposomes

a

AuNPs

DOPC

DOPC/Chol

DOCP

DOCP/Chol

MUA-AuNPs C16-AuNPs C11-Trp-AuNPs C8-AuNPs others

N Y Y N Y

N Y N N N

N N Y N Y

N N N N N

Y: Obvious leakage of CF; N: No obvious leakage of CF.

disruptively perturb the liposome bilayers and induce a very rapid leakage of CF dyes. However, C11-Trp-AuNPs do not lead to obvious leakage of DOPC/Chol liposomes, though the packing of Chol in liposomes would be expected to enhance the binding affinity of C11-Trp-AuNPs to membranes because of the hydrophobic stacking between the aromatic indole group of C11-Trp and the ring-like structure of Chol. Thus, this result highlights the compatibility of the anionic, amphiphilic, and aromatic surface with cholesterol-rich domain on lipid bilayers compared to a merely amphiphilic surface (i.e., C16−AuNPs). To further explore the essential driving force of the membrane−AuNPs interactions, we prepared a new type of liposome using a molecular analogue of DOPC (known as an inverse-phosphocholine lipid),49 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (hereafter referred to as DOCP). DOCP contains a hydrophilic headgroup with an inverted charge orientation relative to DOPC (Figure 1b). The as-prepared DOCP liposomes have a hydrodynamic diameter comparable to DOPC liposomes (∼100 nm) (Figure S7) and a ζ potential of −34.7 mV that is significantly more negative. Liposome leakage assays show that C16-AuNPs display negligible adhesion and interaction with DOCP liposomes (Figure 4a and Table 1). By contrast, C11-Trp-AuNPs induce a gradual leakage of CF dyes within 6 h with a release-kinetics not obviously compromised compared to that in DOPC systems. Moreover, the membrane-disruptive effect of C11-TrpAuNPs disappeared again while filling DOCP liposomes with Chol molecules (Figure 4b and Table 1), like that observed in the DOPC/Chol system. These results suggest that the strong electrostatic repulsion between liposomes and anionic AuNPs

Figure 4. AuNP-induced time-dependent CF leakage from DOCP liposomes (a) and DOCP/Chol liposomes (b) in 20 mM HEPES buffer; [lipid] = 2.3 μM, [AuNPs] = 0.2 nM. Data are presented as mean ± s.d., n = 3. E

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Figure 5. AuNP-induced time-dependent CF leakage from DOPC liposomes (a,d), DOPC/Chol liposomes (b) and DOCP liposomes (c) in 20 mM HEPES buffer; [lipid] = 2.3 μM, [AuNPs] = 0.2 nM. Data are presented as mean ± s.d., n = 3.

In light of the substantially different membrane-disrupting property for MUA-AuNPs, C8-AuNPs, C16-AuNPs and C11Trp-AuNPs, we finally examined and compared the biocompatibility of these anionic AuNPs by monitoring the viability of Hela cells in the presence of different concentrations of these AuNPs. As shown in Figure 6, either C16-AuNPs or

c). This result is consistent with that obtained from anionic AuNPs coated with a monolayer of a binary mixture of 11mercapto-1-undecanesulfonate and OT ligands, which are able to nondisruptively penetrate through cell membranes or lipid bilayers.29 Thus, the results shown in this section strongly imply that the orientation of the surface aromatic indole rings relative to the anionic carboxy of MUA does not affect their exposure and interaction to lipid bilayers. To further examine whether the aromatic indole ring structure on the surface of AuNPs is essential to the membrane−AuNPs adhesion and interaction, new hydrophobic ligands were synthesized by replacing the indole ring group with a phenyl or cyclohexyl group, referred to as Cn-Phe and Cn-Cyh (n = 3, 6, or 11, depending on the ligand length), respectively (Figure 1a). AuNPs protected by MUA mixed with these hydrophobic ligands were prepared and characterized as described above (Figures S12−13). All of these AuNPs are able to induce time-dependent leakage of the DOPC and DOCP liposomes (Figures 5d and S14), similar to that observed for Cn-Trp-AuNPs (n = 3, 6, and 11), and without significant differences in terms of kinetics of CF dye release. Similarly, membrane disruption was not observed in DOPC/Chol (Figure S15). It is particularly worth noting that the membrane-binding capability of anionic AuNPs coated with Cn-Cyh and MUA remains as strong as that of C11-Trp-AuNPs, despite that the surface cyclohexyl groups lack aromaticity. Accordingly, these results strongly imply that the bulkiness of the terminal groups in hydrophobic ligands instead of the aromatic character should be more important to the adhesion and interaction of these anionic and amphiphilic AuNPs with lipid bilayers. Varying the structure of hydrophobic ligands on anionic AuNPs may significantly change the surface monolayer structure or spatial distribution of chemical groups,32 which ultimately distinguishes these surface-aromatic/bulky groupfunctionalized AuNPs from AuNPs coated with MUA and HT/ OT ligands in terms of the different membrane-binding and disrupting capabilities.

Figure 6. Cell viability when treated with different concentrations of AuNPs determined by MTT assay. Results are expressed as mean ± s.d. (n = 3); one asterisk (*) in the same color denotes that cell viability was statistically different in a t-test (P < 0.05).

C11-Trp-AuNPs exhibit higher cytotoxicity compared to MUAAuNPs, highlighting the effect of surface hydrophobicity, which is in parallel to their membrane-disrupting capability. C8AuNPs shows relatively low cytotoxicity, like other anionic amphiphilic AuNPs coated with similar surface ligands reported in the literature,29,32 implying a minimum disruption of cell membranes. Of note, the dose-dependent profile of cytotoxicity for C16-AuNPs and C11-Trp-AuNPs was substantially different. C16-AuNPs were respectively more and less toxic than C11-TrpAuNPs at higher (5 nM) and lower concentrations (0.5 and 2 F

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Langmuir nM). This finding, together with the distinct membranedisrupting property, strongly suggests different mechanisms underlying the cell death arising from the interaction of AuNPs and cell membranes. To verify this, we further developed a dyediffusion assay29 to evaluate the cell membrane-disruption upon the incubation with AuNPs. Cells were incubated with CF (an extracellular tracer dye, 0.01 mM) and AuNPs (2 nM) for 3 h, and then were washed and imaged by fluorescence microscopy. As shown in Figure 7, in contrast to MUA-AuNPs, C16-AuNPs

disruption of lipid vesicles or cell membranes. Moreover, by comparing the results obtained from AuNPs coated with aromatic ligands and cyclohexyl ligands lacking aromaticity respectively, we demonstrated that the bulkiness of the terminal groups in hydrophobic ligands instead of the aromatic character might be more important to the adhesion and interaction of AuNPs with lipid bilayers. The understanding gained through this study thus provides new insight into the mechanism of membrane disruption caused by the hydrophobic interaction between nanoparticles and lipid bilayers. This work is particularly important considering that nanoparticles modified with organic functional molecules possess aromatic and/or bulky groups on the surface, and nanoparticles dispersed in biological fluids may recruit biomacromolecules with these groups exposure to solvents, though the generality of the present paradigm on bilayer disruption remains vague. However, the liposomes being explored herein, as simple analogues to cell membranes, allow more extensive exploration on the nanoparticle−lipid bilayer interaction, which would build more definite correlation between observation on model liposomes and that takes place during the interaction with cell membranes. Interestingly, our results have demonstrated this correlation and that one of the factors causing the cytotoxicity might arise from the disruption of certain cellular membrane domains that are more susceptible to perturbation caused by hydrophobic interactions with nanoparticle surfaces. Moreover, in addition to the overall compositions of ligands on the surface of AuNPs mainly focused in this study, the distribution of surface ligands might also play an important role in the nanoparticle−membrane interactions. This would be a direction meriting further investigation.

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Figure 7. Fluorescence imaging of Hela cells incubated with CF (0.01 mM) and different types of anionic AuNPs (2 nM) in serum-free medium for 3 h at 37 °C; (a) MUA-AuNPs, (b) C8−AuNPs, (c) C16− AuNPs, and (d) C11-Trp-AuNPs. Scale bars: 25 μm.

EXPERIMENTAL SECTION

Materials and Instruments. Hydrogen tetrachloroaute-(III) trihydrate (HAuCl4·3H2O, 99.99%) and dodecylamine (DDA, 98%) were purchased from J&K (Guangzhou, China). Didodecyldimethylammonium bromide (DDAB, 98%), tetrabutylammonium borohydride (TBAH, 98%), 11-mercaptoundecanoic acid (MUA, 95%), and 5(6)-carboxyl fluorescein (CF) were obtained from Acros. Triton X100 and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) were obtained from Sigma-Aldrich. Tetramethylazanium hydroxide (TMAH, 98%) was purchased from Alfa Aesar. 1Hexadecannethiol (HT, 98%) and 1-octylthiol (OT, 98%) were purchased from TCI (Shanghai, China). Phospholipid (DOPC and DOCP) and cholesterol were from Avanti Polar Lipids, Inc. Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Thermo Scientific (Beijing, China). Eppendorf tubes, 96-well flatbottomed plates, and cell culture dishes were purchased from JET BIOFIL (Guangzhou, China). All chemicals were used as received without further purification. All organic solvents were analytical grade at least and used without further purification. UV−vis absorption and fluorescence spectra were recorded using U-3900H spectrophotometer (Hitachi) and F-7000 fluorescence spectrophotometer (Hitachi), respectively. The transmission electron microscopy (TEM) images were taken on a JEOL JEM-1400EX microscope. Malvern nano-900 was used to acquire DLS and ζ-potential data. Synthesis of Surface Ligands. See Supporting Information for details. Synthesis of Au Nanoparticle. DDA-stabilized Au nanoparticles (DDA-AuNPs) were prepared according to a modified procedure reported in the literature.51,52 A 50 mM stock solution of HAuCl4· 3H2O in ethanol was first prepared. The ethanol in HAuCl4·3H2O stock solution (400 μL) was then removed by rotary evaporation, and 2.4 mL of 0.1 M DDAB in toluene was added. The solution was sonicated for 10 min until HAuCl4·3H2O was completely dissolved. Then, 9 mg of DDA in 0.1 mL of DDAB and 25 mg of TBAH in 1.0

and C8-AuNPs, C11-Trp-AuNPs can significantly promote the entry of CF into cytosol. This result thus indicated that AuNPs that are more disruptive to the more negatively charged DOCP liposomes (relative to DOPC) are also substantially more disruptive to cell membranes. Moreover, our results suggested that the cytotoxicity of anionic amphiphilic AuNPs might not rely on the overt disruption of certain domains of cell membranes containing Chol (e.g., lipid rafts). Of note, although assessment from the cytotoxicity indicates that C16AuNPs are substantially more toxic than C8-AuNPs, their capability in inducing CF leakage into cytosol was surprisingly comparable, implying that the cytotoxicity of C16-AuNPs should not result from the overt disruption of membrane permeation of cells. Accordingly, it is very likely that factors contributing to the cytotoxicity of C16-AuNPs are essentially different (despite unknown) to the membrane-disrupting effect of C11-Trp-AuNPs.

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CONCLUSIONS In summary, we have systematically tuned the surface compositions of anionic amphiphilic AuNPs, and explored their interactions with four different types of lipid vesicles. Our results demonstrated the important role of the surface aromatic or bulky groups, relative to the hydrocarbon chains, in the interaction of anionic AuNPs with lipid bilayers. Hydrophobic interaction itself arising from the insertion of aromatic or bulky groups into lipid bilayers is sufficiently strong to result in overt G

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Langmuir mL of DDAB were injected sequentially under vigorously stirring at 40 °C. The reaction was kept for ∼8 h. DDA-coated AuNPs were precipitated by adding an appropriate volume of acetone and washed with acetone once. Finally, the obtained DDA-coated AuNPs were dispersed in 3.5 mL toluene for further use (i.e., for the ligand exchange within 24 h). Surface Ligand Exchanges.31 Oxidized ligands (i.e., disulfide bridged ligands; see Supporting Information) were first reacted with 3fold TBAH for 3 h in a mixture of THF and methanol (1:1) at room temperature to obtain free thiol-bearing ligands. After that, to 3.5 mL as-prepared DDA-AuNPs, a mixture of free thiol-bearing ligands and MUA (1:1) (or MUA alone) dissolved in the mixture of THF and methanol, was injected under vigorous stirring and the reaction was kept for ∼24 h. The precipitation (i.e., the obtained anionic AuNPs) was then washed with toluene, DCM, and acetone sequentially to remove excess chemicals. The residual acetone was completely removed by nitrogen gas purging. Finally, 10 mM TMAH solution (in water) was added to disperse the anionic AuNPs. The concentration of AuNPs was determined using UV−vis spectrometry.53 These AuNPs were also characterized by DLS and TEM. Liposome Preparation. Liposomes were prepared through a wellknown extrusion method.43,45 In a typical experiment (for the preparation of DOPC liposomes), a 25 mg/mL solution of DOPC in chloroform was dried by rotary evaporation at 37 °C to yield thin layers of dried lipid. The residual solvent was removed in vacuum overnight in a desiccator. After that, the lipid films were hydrated in HEPES buffer (pH 7.4, 20 mM) containing 75 mM CF dye. The lipid mixture was kept at room temperature for 1 h with occasional vortex and sonication, and subjected to five freeze−thaw cycles in liquid nitrogen to reduce the size. Then, the lipid mixture was extruded 20 times through a polycarbonate membrane filter (100 nm pore size, Avanti). The unencapsulated CF dye was removed using a 3 kDa centrifugal ultrafiltration filter (Millipore). The obtained liposomes were suspended in HEPES buffer and stored at 4 °C for further use (within 1 week). Malachite green assay was used to determine the concentration of lipids and liposomes.54 CF-Leakage Experiment.43,45 The stock liposome solutions were diluted with 20 mM HEPES buffer to a lipid concentration of 2.3 μM in 1 mL polymethacrylate cuvettes (purchased from Sigma-Aldrich). An aliquot of anionic AuNPs solution was then added into the asprepared liposome solution. The anionic AuNPs induce the release of CF from the liposomes into the surrounding buffer, which results in an increase in fluorescence intensity. We reported the time-dependent leakage as the normalized fraction of released CF given by45

L(t ) =

cells were washed with PBS buffer and cocultured with different concentrations of AuNPs in serum-free DMEM for 24 h. To each well, 10 μL MTT (5 mg/mL) was then added and incubated for another 4 h. The medium was discarded, and 150 μL DMSO was added to dissolve the MTT crystals. The absorbance at 490 nm was measured using a microplate reader (PerkinElmer). To probe the permeability of cell membranes, cells that were seeded in a 12-well plate were cocultured with AuNPs (2 nM) and CF (0.01 mM) for ∼3 h, followed by washing three times with PBS buffers. Then, fluorescence images were recorded using a Nikon Ti-E inverted microscope.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b00035. Synthesis and characterization of surface ligands, and Figures S1−S15 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to acknowledge the financial support of the National Basic Research 973 Program of China (Grant 2014CH932004), the National Natural Science Foundation of China (Grants 21305114, 21375110, and 21475109), the Fundamental Research Funds for Central Universities (Grant ZK1047), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant 21521004), and PCSIRT (Grant IRT13036).

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REFERENCES

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F(t ) − F(0) F(max) − F(0)

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